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[CANCER RESEARCH 37, 1680-1685, June 1977] Early Changes in the Dynamics of Crypt Cell Populations in Mouse Colon following Administration of 1,2-Dimethylhydrazine1 Thomas C. Richards Department of Anatomy, School of Medicine, University of Oregon Health Sciences Center, Portland, Oregon 97201 SUMMARY The effects of the carcinogen, 1 ,2-dimethylhydrazine (DMH), on the proliferative characteristics of the crypt cell population of mouse colon were studied. DMH (20 mg/kg body weight) was injected s.c., weekly, for 2, 8, 16, 20, on 26 weeks. At the end of each treatment period, a group of animals was injected with [3Hjthymidine and killed. After 2 weeks of DMH treatment, the cnypts appeared normal histo logically, but the total number of cells, the number of Ia bebed cells, and the percentage of labeled cells pen crypt column had increased. The relative distribution of labeled cells in crypt columns was not changed. DMH treatment did not affect the phases of the cell cycle of epithelial cells and the transit time of these cells through the crypt. None of the indices of crypt dynamics were altered further with the appearance of focal atypias (aften 16 weeks of DMH). How ever, the total number of cells per crypt increased and the percentage of labeled cells decreased as adenocarcinomas developed in adjacent areas of the mucosa (after 20 to 26 weeks of DMH). The exact robe of these early mucosal changes in the eventual development of malignant tumor has not been established. However, it appears that DMH cancinogenesis may involve two steps: (a) an initial increase in the of mitotically number active cells leading to an en weeks of s.c. injections of DMH (5, 20) and as early as 24 hr after in. instillation of a single large dose of DMH (2). On the other hand Maskens (13) reported an increase in both the total number of cells per crypt and the number of labeled cells pen crypt after 15 weeks of treatment. Localized patho logical lesions (focal atypias) were consistently identified after 12 to 16 weeks of treatment (5, 13, 20); however, a few scattered atypias were noted on the tips of the folds of distal colon as early as 38 days after initiation of treatment (5, 20). Crypt cell populations in the colon are maintained in a dynamic steady state by a balance between the production of new cells (rnitosis) and the loss of old cells (extrusion) (4). Disruptions in crypt dynamics that lead to the development of an abnormal population, the appearance of focal atypias, and eventually neoplastic growth may result from altera tions in (a) the proportion of crypt cells that are mitotically active, (b) the location of mitotically active cells within the crypt, (c) the length of the cell cycle, on (d) the rate of cell bossby extrusion (11). The purpose of this experiment was to study the above indices of cell kinetics after selected periods of weekly s.c. injections of DMH to document the types of pnemabignant changes that may occur in Crypt cell dynamics and to determine whether or not these changes are present prior to the appearance of discrete lesions in the mucosa. bangedcell population; and (b) an eventual transformation of at least some of the crypt cells of the enlarged popula tion. MATERIALS AND METHODS Animals and Treatments INTRODUCTION Several recent reports (5, 6, 20, 21) have shown that a high incidence of adenocancinoma in distal colon can be induced by s.c. injection of DMH2 into mice or rats at weekly intervals for 20 to 30 weeks. However, because of different experimental approaches used by various investigators, a clean understanding of the premabignant changes that oc cur in cobonic mucosa and of the time of their appearance during the induction of cobonic cancer has not emerged. The earliest change noted in the crypt cell population was a doubling of the LI. This was reported to occur after 12 I This work was supported by a grant from the Oregon Division of American Cancer Society. 2 The abbreviations used are: DMH, 1 ,2-dimethylhydrazine; index; i.r., intrarectal; [3H]TdR, tritiated thymidine. Received September 7, 1976; accepted February 23, 1977. 1680 LI, labeling the Female Swiss-Webster mice, at least 60 days old and 20 g in body weight, were housed 5 animals/cage and were given free access to food and water. The animals were given weekly injections of DMH-HCI (20 mg/kg body weight; American Scientific, Portland, Oreg.). DMH was dissolved in 0.001 M EDTA, adjusted to pH 6.5 with sodium bicanbon ate, and injected s.c. Controls were given comparable vol umes of 0.001 M EDTA. Animals were divided into 5 groups with at least 30 expeni mentals and 30 controls in each group. DMH treatment was initiated at the same time for all groups and was carried out for 2, 8, 16, 20, or 26 weeks. One week after cessation of treatment, the animals were killed and their colons were analyzed. Several animals (3 to 7) were used to determine changes in DNA-synthetic activity in cells of the crypt; each animal was given an i.p. injection of [3H]TdR, 0.5 @Ci/g CANCER RESEARCHVOL. 37 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1977 American Association for Cancer Research. Crypt Cell Dynamics and Colon Cancer body weight (specific activity, 20 Ci/mrnole; New England Nuclear, Boston, Mass.), and killed by cervical dislocation 1 hr later. The remainder of the animals were used to deter mine the length of the cell cycle; they were given injection of [3HJTdRand killed at intervals of 1 hr from 2 to 12 hr later or intervals of 2 hr from 12 to 36 hr later. One group was killed at 48 hr. Tissue Handling A segment of distal colon at the level of the pelvic rim was excised and used for analysis because previous studies demonstrated that the descending colon was the most fre quent site of tumor formation (5, 7). Care was taken not to distort the colon during removal. A small section was ex cised from the middle of the segment, washed thoroughly in ice-cold 0.9% NaCI solution, fixed in Camnoy'sfluid for 2 hn, and postfixed in 10% neutral-buffered formalin for 24 hr. The tissue samples were embedded in paraffin and 5-pm sections were prepared. Selected slides were prepared for histological analysis and stained with hematoxylin and eosin. For madioautognaphic processing, 2 slides from each co Ion were treated with 1 N HCI (60°)for 1 hr to hydrolyze the nucleic acids and to remove any free labeled nucleotide (18), immersed in Schiff's reagent, and rinsed in sodium bisulfite (9). The slides were then dipped in NTB3 emulsion (Eastman Kodak Co., Rochester, N. Y.), dried for 1 hn, exposed for 3 weeks, and developed in Dektol (Eastman) (15). Analyses of Crypt Columns Crypt columns, defined in these experiments as columns of epithelial cells along 1 side of a mucosab crypt, were selected for cell kinetic analysis if they were: (a) not dis tonted by the presence of abnormal cell lesions (focal atyp ias) or tumors, (b) sectioned longitudinally from top to bottom, and (c) in contact with the musculamis mucosae. The sections selected for examination were spaced so that each crypt column was encountered only once. Fifty crypt columns were analyzed for each animal. LI. Four indices of kinetics of crypt cells were measured. The length of the crypt column was determined by counting the cell positions starting from the base of the crypt and continuing up to the junction with surface cells (Chart 1; Ref. 12). A cell was considered labeled if 3 or more grains were seen over the nucleus. The number of labeled nuclei in each cnypt was recorded. The LI was determined by calcu bating the ratio (3). @ Labeledcells in a population Total cells in a population @ Student's t test was used for comparisons between means of values from DMH-tmeated and control animals (19). Absolute and Relative Distribution of Labeled Cells In Crypt Columns. The length of each crypt was normalized to 100 units because of the variations in crypt length in ani mals from different treatments. For a determination of what portion of the crypt contained the mitotically active cells, Control 2 week DMH treatment 26 week DMH treatment Chart 1. Distribution of proliferating cells in crypt columns of colons from control animals and animals treated with DMH for 2- and 26-week, periods. Dark cells, cells that are labeled 1 hr after injection with [3H]TdR. Changes in the distribution of mitotically active cells were determined by counting the numbers of labeled cells in quarters and segments of the crypts. Division of the crypts into quarters and segments was based on the relative lengths of the crypt column. crypts were divided into quarters, and each labeled cell was assigned to a specific quarter. An analysis of variance was used to test for differences in values within each quarter of the crypt. The Newman-Keuls multiple range test was used to determine differences in values between the quarters of the crypt. Differences between the means from control and DMH-treated animals for each quarter were determined by an analysis of variance. The crypt was further divided into 10 equal segments to localize more accurately changes in the distribution of mi totically active cells; the percentage of labeled cells in each crypt segment was calculated to determine whether on not there was an increase in the proportion of cells that were dividing. These data were transformed for statistical pun poses by the arc sine of the square mootof the percentage, and differences in distribution of labeled cells between con tnol and DMH-tneated animals were determined by covani ance analysis (19). Length of the Cell Cycle. The phases of the cell cycle of crypt cells were determined from a curve that was gener ated by plotting the fraction of labeled mitoses against the time after injection with [3H]TdR (17). At least 50 mitoses from each animal were counted. The curves for the control and DMH-tmeatedanimals were fitted to the points by hand, and individual pains of points along the curves were corn pared statistically by Student's t test. Rate of Migration In Crypt Columns. The velocity at which cells moved upward in the crypt was deduced from the distribution of mitotic cells in the crypt. The number and position of all mitotic figures were recorded . It was assumed that the length of mitosis was 1 hr at all levels of the crypt. The rate of migration of cells at any position in the crypt was considered to be equal to the cumulative rate of prolifera tion up to that position. The mateof proliferation was deter mined by the mitotic index of each position (1). Proportion of cells in a population Mitotic time that are mitotic RESULTS Gross and Microscopic Findings. The most striking result observed in colons from animals treated with DMH for 2 JUNE 1977 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1977 American Association for Cancer Research. 1681 T. C. Richards weeks was an increased cellularity (compare Figs. 1 and 2). Although only the changes in the crypts were quantitated, there appeared to be an increase also in the number of cells in the larnina pmopnia and submucosa. Crypt cell length initially increased by about 25% over control values (Table 1). Subsequent treatments caused no further change in the length of the crypts until after 26 weeks when the crypt length increased by 40% oven controls. This marked in crease in the total number of cells coincided with the ap pearance of frank tumor in adjacent areas of the mucosa. Focal atypias were first observed in scattered crypts after 16 weeks of DMH treatment (Fig. 3). The atypias, defined as localized lesions of atypical cell structure and high mitotic activity (5), were usually located in the outer or lurninab halves of the cnypts. The lesions were irregular in shape and occupied areas of the larnina propnia; they were not ob served deep to the musculanis mucosae. A few small but grossly visible tumors were observed in the colons from some animals after 20 weeks of DMH treat ment. Multiple barge lesions were observed throughout the distal colon of almost all animals treated for 26 weeks with DMH (Fig. 4). In many of the tumors examined histobogi cabby,cells in the base of the lesion had invaded the submu cosa; this was interpreted as evidence of cancer. Lange ovemgrowths of cells blocked the lumens of many colons examined. Lb. The mean number of labeled cells per crypt column and the LI increased after 2 weeks of DMH treatment 1). The number of labeled cells pen crypt column (Table approxi mately doubled compared to control values, whereas the LI increased by a lessen amount. Neither of these values changed further with subsequent treatment until frank tu Table 1 Effects of DMH treatment on cells of crypt columns from distal colon Total number of cells (crypt length), number of labeled cells, number of mitotic cells, and percentage of labeled cells per column of crypt cells in histologically normal cryptsfrom control and DMH treated [3H]TdR.Length animals killed 1 hr after i.p. injection of of treat ment @ Crypt length 37.9 ±17b crypt 7@7 0.7 LI 20.5 31.3 ±1.2 p<0.01 4.0 ±0.6 12.9 DMH (4) 39.4 ±1.8 8.5 ±0.9 21.8 Control 28.3 ±0.7 3.8 ±0.9 13.7 21.2 15.2 Control (5)@ p<0.018 @ (5) p<0.0116 p<0.01 DMH (3) Control (3) p<0.0120 42.6 ±1 .8 29.0 ±1 .4 p<0.01 9.0 ±0.3 4.4 ±0.8 17.726 DMH (3@ 42.1 ±0.5 7.4 ±0.3 DMH (7) Control (7) 52.7 ±1.5 31.8 ±1.0 7.2 ±0.4 3.5 ±0.2 p < 0.005 p < 0.01 b Mean C Controls 1682 0.01). Although there was some variation between the treat ment periods as to the number of labeled cells within each of the quarters, analysis of variance revealed that these differences were not significant (p > 0.05). The analysis of variance and the Newman-Keuls multiple range test showed that there were significantly greater numbers of labeled cells in Quarter I compared to Quarter lb (p < 0.01) and in Quarter II compared to Quarter III (p < 0.01) for all durations of treatment. The effects of treatment on LI in individual segments of the crypt columns are shown in Chart 3. Covaniance analy sis of data from control and DMH-treated animals indicated that the slopes of the 2 lines were the same but that theme was an overall increase in LI in the expemimentals(p < 0.01). The distribution of labeled cells from animals treated for 26 weeks was different than that from animals in other treat ment groups. The results revealed that (a) DMH treatment increased the number of labeled cells uniformly throughout the proliferative zone of the crypt; and (b) although the crypt columns lengthened initially, the relative distribution of the labeled cells in the cnypts was not altered by DMH treatment until frank tumor development after 26 weeks. In these later animals, relatively fewer cells were seen at the base and relatively more cells were seen at the mouth of the crypt. Length of the Cell Cycle. The lengths of the cell cycle and S phase (DNA synthesis time) were measured in the crypt cell populations at selected times after DMH treatment (Chart 4). The values from animals treated with DMH for various durations were not different; therefore, the values Labeled cells/ (wk) (%)2 Treatment DMH(4) a Numbers momsbegan to develop after 20 to 26 weeks. In crypt cob umns adjacent to developing tumor, the crypt lengths in creased markedly, whereas comparable increases in the number of labeled cells did not occur; hence, the LI de creased in these crypts. Absolute and Relative Distribution of Labeled Cells in Crypt Columns. The distribution of labeled cells in each quarter of the Crypt column is shown in Chart 2. The abso lute numbers of labeled cells in each quarter of the crypts were greaten in DMH-tneated animals than in controls (p < in parentheses, number 13.7 11.4 this group were mishandled of animals. and are not Weeksoft8IG2O26 Treatment @@I62O@6 First included. té162026 Second Quarters ± SE. in j:: 2eI62O@6 Third of the crypt Fourth column Chart 2. Distribution of the number of cells in quarters of the crypt that were labeled 1 hr after i.p. injection of [3H]TdR. Each point Is the mean; bars, SE.; ----, control animals; —, DMH-treated animals. CANCER RESEARCHVOL. 37 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1977 American Association for Cancer Research. Crypt Cell Dynamics and Colon Cancer showing that treatment of mice with DMH for 26 weeks induces a high incidence of adenocarcinoma in mucosa of distal colon. Small lesions, consisting of clusters of imnegu lamlyshaped crypt glands lined with atypical epithelial cells (focal atypias), appear on the mucosal folds of colon prior to the development of frank tumor. These lesions commonly U occur between the 12th (5, 20) and 15th to 16th (13) weeks 0 of DMH treatment, although a few scattered atypias were noted after 38 days of treatment (5, 20). In this study, atypias were first observed in colonic mucosa after 16 weeks of DMH treatment. In addition, the present results demon strate that DMH leads to a marked increase in cellularity in colonic cnypts several weeks before the appearance I segmentsof crypt column Chart 3. Distribution of the percentage of cells that are labeled 1 hr after i.p. injection of [3HJTdRin segmentsof crypt columnsfrom control animals (x- — - x) and DMH-treated animals after 2, 8, 16, and 20 weeks of treatment (x—x) and after 26 weeks of treatment ( 0). Each point is the mean; bars, SE. long before the appearance Time (hours after [@@-Thymidineinjection) Chart4. Lengthof the cell cyclesandthe S phase(DNAsynthesistime)of the crypt cell populations. Each point is the mean; bars,S.E.; - - - -, control animals; —, DMH-treated animals. for all DMH-tneated groups were combined. The S phase was 7 to 8 hn, and the length of 1 cell cycle was 25 to 26 hr for crypt cells of colons from both control and DMH-tneated animals. There was no significant effect of treatment on these indices. Rate of Migration In Crypt Columns. Estimates of the maximum rates of cell migration along the crypt column by cells in the nonprolifenative compartment of the crypt were 1.1 and 0.7 cell positions/hr for DMH-tneated and control animals, respectively. This difference reflects the increased number of mitotic cells per crypt after DMH treatment. However, because the crypt length also increased after DMH treatment, the cells had to migrate a greater distance in the crypt before reaching the mouth of the gland. As a result the cell transit times through the nonproliferative zone of the crypt (i.e. , Quarter IV) were comparable in expenimentals and controls (9 and 10 hn, respectively). DISCUSSION The results of the present experiments confirm the work of Thumnhemret al. (20), Deschner (5), and Maskens (13) in JUNE 1977 of atyp ias. The proportion of cells in the elongated crypts that were actively dividing, i.e. , cells in mitosis or in the S phase of the cell cycle, also increased, although the relative distnibu tion of dividing cells within the crypt was not affected. Further changes in the indices of cell kinetics of mucosal crypts were not observed until frank tumor had developed after 26 weeks of DMH treatment. These findings suggest that the mechanisms that regulate crypt dynamics are “me set―and that a bangercrypt cell population is established of adenocarcinoma. It cannot be determined from the present results whether these initial changes are an integral part of the carcinogenic process or represent a less specific response to DMH. Chan et al. (2) reported that, following a single in. instilla tion of DMH, the LI in distal colon increased by 2-fold after 24 hr and by 3-fold after 2 weeks. These changes in the LI occurred earlier and were greaten in magnitude than those observed here. Such differences in latency and amplitude of response of DMH-sensitive cells in colon may be due to the route of administration and/orthe concentration of DMH. The eventual development of neoplastic growth in mu cosa of colon may be dependent in part on disruptions in the normal distribution of dividing cells in the crypt. Epithe hal cells located in the bower two-thirds of the cobonic crypt (proliferative zone) undergo DNA synthesis and proliferative activity (4). These cells migrate toward the cryptal lumen and cease dividing as they reach the upper portion of the crypt (4, 12). Thumnhemm et a!. (20) and Lipkin (10) reported that, in animals treated with DMH for 12 weeks, a greater number of dividing cells were located in cell positions fur them from the crypt base than in comparable controls. This alteration was interpreted as evidence for the expansion of the proliferative zone of the crypt. The results of the present study support this interpretation in that an absolute en langement of the proliferative zone was observed; i.e., more cell positions in the crypt columns contained mitotically active cells. However, a relative enlargement of the prolifem ative zone was not demonstrated because the total number of cells in the crypt column also increased. Thus, the dis tance between the highest cells undergoing division and the mouth of the crypt remained fairly constant. Therefore, the action of DMH does not alter the relative distribution of labeled cells in crypts, at least not during the inductive period prior to the appearance of tumors. This difference in interpretation between the present study and the work of Thunnhemret a!. (20) and Lipkin (10) may be a reflection of the method by which the crypt cells were analyzed. In this study the LI was calculated from populations of cells in the 1683 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1977 American Association for Cancer Research. T. C. Richards quarters and segments of crypt columns, and these popula tions were normalized to reflect fluctuations in the total number of cells in the crypt column. In contrast, Thumnhemn et at. (20) and Lipkin (10) determined the Lbof individual cell positions and did not take into account the crypt length. The present results show that the crypt columns are en bangedby 2 weeks after initial DMH treatment. Chang and Nadlem(4) suggested that the balance between cell produc tion and cell loss is dependent in part on the maturity of the experiments. However, it appears that the DMH-induced transformation to tumor occurs in an altered population of crypt cells. ACKNOWLEDGMENTS I gratefully acknowledge the technical assistance of Patricia Richardson and the expertadviceof Dr. H. Weitlauf. cells. An increased crypt length might be expected to result from an increase in cell division because less mature cells would be located higher in the crypts and would not be lost until they had had time to mature. Thus, lengthening of the crypt would continue until sufficient time for maturation had elapsed, at which point cell bossshould again equal cell production (14). Under these conditions the population size would be reset at a higher bevel. Kanagalingam and Babis (8) postulated that the develop ment of tumor results from a reduced ability of cells to repair DNA damaged by chronic exposure to high doses of a carcinogen. Colonic mucosa consists of cells with a short G phase (10 hr) and a life-span of 100 to 120 hr (3, 4). In such a tissue errors in DNA could be incorporated into the next generation of cells if DNA repair is not completed before cell division occurs. In this way the errors would accumulate during chronic exposure to the carcinogen. Enlargement of the population may play a significant mole in REFERENCES 1. Cairnie,A. B., Lamerton,L. F., andSteel,C. G.CellProliferationStudies in the Intestinal Epithelium of the Rat. I. Determination of the Kinetic Parameters. Exptl. Cell Res., 39: 528-538, 1965. 2. Chan, P. C., Cohen, L. A., Narisawa, T., and Weisburger, J. H. Early Effects of a Single Intrarectal Dose of 1,2-Dimethylhydrazine in Mice. Cancer Res., 36: 13-17, 1976. 3. Chang, W. W. L., and Leblond, C. P. Renewal of the Epithelium in the Descending Colon of the Mouse. I. Presence of Three Cell Populations: Vacuolated-columnar,Mucousand Argentaffin.Am. J. Anat., 131:73100, 1971. 4. Chang, W. W. L., and Nadler, N. J. Renewal of the Epithelium in the Descending Colon of the Mouse. IV. Cell Population Kinetics of Vacuo lated-columnarand MucousCells.Am. J. Anat., 144:39-56, 1975. 5. Deschner,E. E. ExperimentallyInducedCancerof the Colon. Cancer, 34: 824—828, 1974. 6. Druckrey, H. Production of Colonic Carcinomas by 1,2-Dialkylhydrazines and Azoxyalkanes. In: W. J. Burdette (ed), Carcinoma of the Colon and Antecedent Epithelium, pp. 267-279. Springfield, Ill.: Charles C Thomas, Publisher,1970. 7. Haase,P.,Cewen,D. M., Knowles,J. C., andCooper,E. H.Evaluationof Dimethylhydrazine Induced Tumours in Mice as a Model System for Colorectal Cancer. Brit. J. Cancer, 28: 530-543, 1973. the carcinogenic response to DMH by increasing the num ben of cells at risk. Lipkin and Deschnem (11) proposed that the inductive period represents the time necessary for enough generations of cells to pass until a population is reached that possesses the characteristics conducive to malignant growth. Under the conditions are required before transformation in mouse skin that DNA and RNA synthesis â€ãnd that actual tumor formation is increased by is effected by specific ‘ ‘promoters'â€ãcting on the altered cells. That a similar mechanism may be involved in DMH-induced colon cancer is suggested by the finding that, after the initial changes in LI, no further alterations were observed in the crypt until the appearance of focal atypias after 16 weeks of treatment. Thus, the action of DMH may require 2 steps: (a) an initiating step that stimulates alterations requisite hal Cells in Diseases Leading to Colonic Cancer. Cancer, 34: 878—888, 1974. 11. Lipkin, M., and Deschner,E. Early ProliferativeChangesin Intestinal Cells. Cancer Res., 36: 2665-2668, 1976. 12. Lipkin, M., and Quastler, H. Cell Population Kinetics in the Colon of the Mouse. J. Clin. Invest., 41: 141-146, 1962. zine-induced Rat Colon Adenocarcinoma. Cancer Res., 36: 1585-1592, 1976. 14. Maurer, H. R. Chalones: Specific Regulators of Eukaryote Tissue 15. 16. 17. 18. a pool of DMH-sensitive cells to divide and (b) a promoting step in which the en barged cell population requires chronic insult in order to undergo pp. 270-271. NewYork: McGraw-HillBook Company,1965. 10. Lipkin, M. Phase1 and Phase2 ProliferativeLesionsof Colonic Epithe 13. Maskens,A. P. Histogenesisand Growth Patternof I ,2-Dimethylhydra Neoplastic transformation in colon may be similar to the 2-stage induction of skin tumors. O'Brien etal. (16) showed “ initiators' Damage by Alkylating Agents. Cancer, 36: 2364-2372, 1975. 9. Lillie, R. D. Histopathologic Technic and Practical Histochemistry, Ed. 3, of the present study, the results suggest that 140 to 175 generations of cells, or somewhat less because all proliferating cells may not recycle immediately, occu ns. 8. Kanagalingam,K., and Balis,M. E. In viva Repairof RatIntestinalDNA for malignant changes. The significance of the early mucosab changes to the oncogenic process cannot be determined from the present 19. Growth. In: G. P. Talwar (ed), Regulation of Growth and Differential Function of Eukaryote Cells, pp. 69-77. New York: Raven Press, 1975. Meissier, B., and Leblond, C. P. Preparation of Coated Radioautographs by Dipping Sections in Fluid Emulsion. Proc. Soc. Exptl. Biol. Med., 96: 7-10, 1957. O'Brien, T. G., Simsiman, R. C., and Boutwell, R. K. Induction of the Polyamine-biosynthetic Enzymes in Mouse Epidermis by Tumor-promot ing Agents. Cancer Res., 35: 1662-1670, 1975. Quastler, H., and Sherman, F. G. Cell Population Kinetics in the Intes tinal Epithelium of the Mouse. Exptl. Cell Res., 17: 420-438, 1959. Rogers, A. W. Techniques of Autoradiography, pp. 207-222. Amsterdam: Elsevier Publishing Company, 1967. Steel, R. G. D., and Torrie, J. H. Principles and Procedures of Statistics, pp. 156-158.NewYork: McGraw-HillBook Company,1960. 20. Thurnherr, N., Deschner, E. E., Stonehill, E. H., and Lipkin, M. Induction of Adenocarcinomas of the Colon in Mice by Weekly Injections of 1,2Dimethylhydrazine. Cancer Res., 33: 940-945, 1973. 21. Weisburger,J. H. Colon Carcinogens:Their Metabolismand Mode of Action. Cancer, 28: 60-70, 1971. Fig. 1. Section of mucosa of colon from control animal. The nuclei of the crypt columns are more basophilic than those in Fig. 2. H & E, x 60. Fig. 2. Sectionof mucosaof colon from animaltreatedwith DMHfor 2 weeks.Notethe elongatedcrypt columnsand increasedcellularityof the lamina propriacompared tothatinFig.1.H & E, x60. Fig. 3. Section of mucosa of colon from animal treated with DMH for 16 weeks. Note the focal atypia at the tip of the mucosal fold. H & E, x 60. Fig. 4. Section of mucosa and tumor of colon from animal treated with DMH for 26 weeks. This particular intramucosal carcinoma had not invaded the submucosa. Goblet cells, well demonstrated in the adjacent mucosa, are markedly reduced in the tumor. Periodic acid-Schiff, x 60. 1684 CANCER RESEARCH VOL. 37 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1977 American Association for Cancer Research. @ @ ;@r@ :.@ k@1 .-@. .,@*. @r(•1 @ ‘.>5@: @ -_..-‘@,r.';_@ @ . ‘i.:@: @ @. :@ - , . ... .. . _.,_v. -@ - . @ @ - *,_p. .@ .-. . i. ‘ @ ,@ @4‘?@@ B, •@Lz;@@.-@ @ IP$:. @: @ ‘- @ - . @, @-.. ,@ ,..1,.-. @ @ .. ;-@:.@L---'.'@•' . ‘@ .: , ‘ . __@@;4@ @ .@. @ tV • @ @ ., @.:-.-@@-‘@‘ @ ,, .@: @ ... ,,. . • ;@ .@., . @@‘tJf ,@ @ 1@.. . .. -@ ‘ .. @“. @ . @ @ @ @ . • . @-@: .. #.@‘.- . ‘,. . ; . @ , - - . .@.;_ :@ .. :--@ @‘:-@ @ .@; ‘.‘ @ ,... . ‘ ...-@ .;@ . . ., I : ,& , , . , - @-. - . JUNE 1977 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1977 American Association for Cancer Research. 1685 Early Changes in the Dynamics of Crypt Cell Populations in Mouse Colon following Administration of 1,2-Dimethylhydrazine Thomas C. Richards Cancer Res 1977;37:1680-1685. 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